Hyperhomocysteinemia is an independent risk factor for myocardial infarction and stroke, yet the mechanisms by which homocysteine (Hcy) promotes arteriosclerosis are not clear. Most of the reported biological effects of Hcy in vascular cells have been attributed to oxidative mechanisms, which were observed at Hcy concentrations higher than 1 mM or higher, and can be mimicked by cysteine, another nonpathogenic biothiol. Thus, a biochemical mechanism unique to Hcy remains to be identified. We have proposed hypomethylation as a specific mechanism by which Hcy induces vascular injury and leads to cardiovascular disease. The basic hypothesis of the ongoing and this proposed projects is that Hcy, at clinically relevant concentrations, selectively inhibits EC growth through a hypomethylation-related mechanism. The ongoing research is designed to investigate the role of Ras demethylation in Hcy-EC growth, to dissect the mechanism in Hcy signaling using cellular and animal models. Because damage to EC is a key feature of arteriosclerosis, the growth inhibition of EC may represent an important mechanism to explain Hcy-induced arteriosclerosis. In the proposed study, we hypothesize that hypomethylation of other molecules may also play an important role in Hcy-related EC growth inhibition. We added two new aims to characterize methylation status of genomic DNA and protein, to examine the activities of histone methyltransferase in Hcy-treated EC (Aim 4), and to identify new functional target genes using retrovirus-mediated genetic screening and radiolabelled methylation sensitive two-dimensional electrophoresis proteomics (Aim 5). These two new aims are expansion of the funded project and would explore key functional molecular mechanisms by which Hcy inhibit EC growth. The broad, long-term objective of this proposal is to elucidate Hcy signaling in EC growth inhibition, and to evaluate its importance in the role of atherogenesis in Hcy pathology. If we can identify the key events in Hcy-induced arteriosclerosis, genetic or biochemical approaches to block these steps could lead to therapeutic advantage.